Process for obtaining aroma components and aromas from their precursors of a glycosidic nature, and aroma components and aromas thereby obtained

ABSTRACT

A method is disclosed for obtaining aroma components and aromas from their glycosidic precursors containing a β-apioside with the use of β-apiosidase.

This is a continuation of application Ser. No. 07/681,520, filed asPCT/EP90/01545 Sep. 7, 1990, now abandoned.

The present invention relates to a process for obtaining aromacomponents or aromas from their precursors of a glycosidic nature, aswell as to the aroma components and aromas obtained by this process.

TECHNICAL BACKGROUND

In some wine varieties, such as muscats, the aroma compounds exist intwo forms, free and bound. The free fraction consists of odoriferousvolatile substances, chiefly terpenols. The bound fraction containsprecursors of terpenols, especially non-odoriferous diglycosides, formedfrom α-L-rhamnopyranosyl-β-D-glucopyranosides (designated Rha-Glc), fromα-L-arabinofuranosyl-β-D-glucopyranosides (designated Ara-Glc) and fromβ-D-apiofuranosyl-β-D-glucopyranosides (designated Api-Glc), in whichthe glucopyranose provides the link between the terpene residue(designated Terp) and the disaccharide, according to the formulae:

    Rha(1→6)Glc-Terp

    Ara(1→6)Glc-Terp

    Api(1→6)Glc-Terp

The aroma fraction in the form of precursors is most often much largerthan the free aroma fraction (typically by a factor of 3 to 10), and itcan reach high concentrations, of the order of a few milligrams perliter.

Taking into account, in addition, the particularly low threshold ofolfactory perception and the aromatic quality of terpene alcohols, thereis, in these vine varieties, a most important unexploited aromapotential.

The terpene glycosides, present in the juice, may be hydrolysed usingcommercial enzyme preparations with a wide variety of specifications.The enzymatic liberation of terpenols, which reflects the free naturalaroma of the fruit more faithfully than that revealed by thermalhydrolysis at the pH of the juice, is hence possible; however, thecontrol of this liberation for an industrial exploitation of the aromapotential presupposes that the glycosidases responsible for thehydrolyses are defined and their mechanism of action established.

BACKGROUND ART

Strauss et al. (in Parliament et al., Biogeneration of aromas, 1986,American Chemical Society, Washington, D.C., pp. 222-239) discuss theimportance of monoterpenes in grape and wine flavor and the extent towhich these compounds contribute to varietal character which isespecially significant in winemaking.

Pisarnitskii, A. F. (Chemical Abstract 128650c, 1971, Columbus, Ohio)discloses a method of imparting odors to wines via the enrichment of theproduct with substances in the pulp to increase the activity of pulpenzymes that are responsible for enzymatic synthesis of the aromaticsubstances present in the product and to impart the desired arome to thefinished product.

Drawert, F. et al. (J. Agric. Food Chem., 1978, vol. 26, no. 3, pp.765-766) disclose the finding of the monoterpenes citronellol, linalooland geraniol in the oderous constituents produced by the yeastKluyveromyces lactis in aerobic submersed culture. By changing of theculture conditions, it was possible to influence the biosynthesis ofcitronellol in K. lactis.

SUMMARY OF THE INVENTION

The present invention is based on a demonstration of the mechanism ofenzymatic hydrolysis, such mechanism being of the sequential type.

Thus, the process according to the present invention for obtaining aromacomponents and aromas from their precursors of a glycosidic nature ischaracterized in that:

in a first stage, an enzymatic hydrolysis of a glycosidic substratecontaining at least one of the said precursors is performed with atleast one enzyme chosen in accordance with the structure of the saidprecursor, to liberate the corresponding monoglucosides by cleavage at aglycoside bond;

in a second stage, an enzymatic hydrolysis of the product of the firststage is performed with at least one enzyme other than or identical tothat/those of the first stage and designed to liberate the aromacomponents and aromas by cleavage of the aglycone-carbohydrate linkbond.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Chromatographic profile of α-rhamnosidase and β-glucosidaseactivities.

FIG. 2: Chromatographic profile of α-arabinosidase, α-rhamnosidase andβ-glucosidase activities.

FIG. 3: Chromatographic profile of α-arabinosidase, α-rhamnosidase andβ-glucosidase activities following DEAE-Sepharose CL-6B ion exchangechromatography.

FIG. 4: Chromatographic profile of α-arabinosidase activity followingconcanavalin A-Ultrogel affinity chromatography.

FIG. 5: Chromatographic profile of α-arabinosidase activity as afunction of pH and temperature. FIG. 6: TLC monitoring of the enzymatichydrolysis of synthetic Ara-Glc-pNP and Rha-Glc-pNP substrates.

FIG. 7: TLC monitoring of the enzymatic hydrolysis of a glycosidicextract.

FIG. 8: Monitoring by GC of the enzymatic hydrolysis of a glycosidicextract by α-arabinosidase±β-glucosidase.

FIG. 9: Monitoring by GC of the sequential hydrolysis of a glycosidicextract by α-rhamnosidase+β-glucosidase.

FIG. 10: HPLC chromatogram of terpene glycosides.

FIG. 11: GC Chromatograms of glycosidic extracts from the must of Muscatde Frontignan grapes (A); enzymatically treated with Hemicellulase (B);enzymatically treated with Klerzyme 200 (C).

FIG. 12: GC Chromatograms of glycosylated aroma fractions from the mustsof naturally sweet wines: (A) enzymatically treated; (B)non-enzymatically treated; and dry wines: (C) enzymatically treated; (D)non-enzymatically treated.

DETAILED DESCRIPTION OF THE INVENTION

"Substrate" is understood to mean any substance containing a precursorof a glycosidic nature of an aroma component or aroma.

By way of an example, the glycosidic precursor may be a vegetablematerial derived from grapes, such as grape juices, wines and theirderivatives, for example all drinks containing wine, grape juice or arelated substance, as well as by-products of the vinification ofaromatic vine varieties, especially muscats. The four components of theenzyme system needed for the hydrolysis of the terpene glycosides ofgrapes to liberate terpenols, odoriferous volatile substances, are nowknown according to the invention: an α-arabinosidase, an α-rhamnosidaseand a β-apiosidase for the first stage, and β-glucosidase for the secondstage. The work leading to the present invention has enabled, in effect,to be concluded that the hydrolysis of terpene diglycosides does notproceed from the action of a single glycosidase but, on the contrary ofseveral, operating in pairs according to the following two-stagesequential mechanism:

Stage 1: Action of an α-arabinosidase, an α-rhamnosidase and aβ-apiosidase liberating the corresponding terpene monoglucosides bycleavage at the (1→6) glycoside bond.

Stage 2: Action of a β-glucosidase providing for the liberation of theterpenols by cleavage of the terpene aglycone-carbohydrate link bond.##STR1##

The last key enzyme, namely the β-glucosidase, on which the finalliberation of the terpenols depends, preferably exhibits minimalinhibition by glucose, activity at acid pH values and high affinity withrespect to the aglycones engaged in the glycosidic substrates, in orderfor the proposed process to possess complete efficacy in itsapplications.

According to the invention, the substrates (Ara-Glc-pNP, Rha-Glc-pNP,Api-Glc-pNP, Ara-pNP, Rha-pNP, Api-pNP, Glc-pNP) enabling the activitiesto be measured, have also been defined, these conditions being essentialfor the production of the corresponding enzymes.

The process according to the present invention is exemplified by theexploitation of the aroma potential of grapes, the primary plantmaterial being chosen from grape juices, wines and derivatives, as wellas by-products of the vinification of aromatic vine varieties,especially but not exclusively muscats.

However, the present invention is not limited to this application. Ineffect, the mechanism demonstrated is a general mechanism leading toaroma components and aromas which are not necessarily terpenic, andwhich is applicable to all plant products other than grapes: fruits,fruit derivatives (for example drinks) and fruit by-products; aromaticplants and flowering plants, as well as derivatives and by-products ofthese plants; other plants such as tea and tobacco; and even plantmaterial originating from in vitro cell cultures; with the followingprovisos:

(a) that these plant products contain, in sufficient amounts, aroma orperfume precursors of a glycosidic nature, including terpenols andglycosides other than those encoutered in grapes,

(b) that the specific glycosidases correspond to the structure of theglycosidic precursors; and

(c) that the natural medium does not contain inhibitors of the enzymesapplied.

As fruits containing glycosides, other than grapes, which can be used inthe process of the present invention, apricots, mangos, papayas andpassion fruit may be mentioned, inter alia. As a flowering plant, therose may be mentioned inter alia.

It is also possible to use a glycosidic extract as a glycosidicsubstrate, or the hydrolysis can alternatively be performed on a naturalmedium containing the precursors.

Thus, a large number of possibilities are offered for carrying out theprocess according to the present invention:

working on a natural medium, as described above, the bound aroma isliberated, thereby increasing the aroma of the product itself;

it is also possible to treat a given glycosidic extract (for example ofpapaya) and to introduce the aroma obtained into another product, forexample a drink (grape juice);

it is also possible to introduce one (or more) glycosidic extract(s)(for example papaya extract, marc extract) into a liquid substrate (forexample grape juice, natural drinks) and to apply the process accordingto the invention, thereby liberating the aroma in situ. It is alsopossible to contrive that the aroma is liberated later, at the desiredmoment, in a food, a drink or a perfume.

The term "enzyme" defines here any means capable of obtaining thecorresponding enzymatic activity. The enzymes employed in the processaccording to the present invention can be of any origin: bacterial,fungal, yeast, plant, animal or synthetic. Enzymes produced by geneticmanipulations using host microorganisms are also encompassed accordingto the invention.

Microorganisms can thus be modified by techniques known to those versedin the art for producing an enzyme or several enzymes, which can beusable in the process.

The process of the invention makes it possible to obtain, in particularas aroma components or aromas, terpenols such as geraniol, linalool,hydroxylinalools, oxydes of linalool, nerol, citronellol, α-terpineol,norisoprenoids compounds (such as hydroxy-3-damascone, 3-oxo-α-ionol),terpene polyols and alcohols such as phenyl ethyl and benzyl alcohol, orthe like.

According to the invention, it is also possible, depending on theprofile of the aroma bound to the precursors, which can be differentfrom the free aroma, or depending on the specificity of the enzyme used,to envisage the production of a novel aroma.

Knowledge of the mechanism results in the production of enzymes havingnovel characteristic properties and technological capabilities.

According to a particular embodiment of the process of the presentinvention, corresponding to the case where the substrate contains onlymonoglucosides, the enzymatic hydrolysis is performed directly withoutpassing through the first stage.

In the definition of the invention, as mentioned above, it was statedthat the process involved a two-stage hydrolysis. However, the inventionis in no way limited to the use of two stages with successive additionsof separate enzymes. As a variant, it is in effect, perfectly possible,especially if it is the same enzyme or enzymes which liberate(s) themonoglycosides and also the aroma components and aromas, to bring theglycosidic substrate into contact in a single stage with the chosenenzyme or enzymes, the enzymatic hydrolysis then proceeding in at leastone reaction phase leading to the desired aromas and/or aromacomponents. This embodiment of the process of the invention can provesuitable, for example, if at least one microorganism encoded forproducing the enzyme or enzymes useful in the reaction is used for thereaction with the glycosidic substrate. In the description whichfollows, when a first stage and a second stage are referred to in theinterest of the convenience of the description, this simply means thatthe hydrolysis reaction proceeds in several phases, but this in no waymeans that these reaction phases necessitate separate additions ofenzymes.

The following examples are provided as a means of illustrating theinstant invention, and are not intended to limit the invention.

EXPERIMENTAL SECTION

A. SUBSTRATES

Among the substrates used, p-nitrophenyl β-D-glucopyranoside (Sigma,U.S.A.), p-nitrophenyl α-L-arabinofuranoside (Sigma, U.S.A.) andp-nitrophenyl α-L-rhamnopyranoside (Extrasynthese, France) arecommercially available. Geranyl β-D-apiofuranosyl-β-D-glucopyranoside isan extract of the medicinal plant species Hypoxis acuminata. The otherglycosides were synthesized: their synthesis employs three stages,starting with the corresponding peracetylated saccharide.

The first stage consists in the activation of the anomeric carbon of theterminal carbohydrate group of the peracetylated correspondingsaccharide, by introduction, onto this carbon, of a halogen such aschlorine or bromine, or an imidate such as trichloroacetimidate.

Thus, hexaacetyl-α-chlororutinoside is obtained by the action of zincdichloride and dichloromethyl methyl ether on peracetylated rutin in aninert solvent such as chloroform or methylene chloride, under anhydrousconditions.

Tetraacetyl-α-bromo-D-glucopyranoside and hexaacetyl-α-bromorutinosideare prepared by the action of gaseous hydrobromic acid onpentaaceto-D-glucopyranose and on heptaacetorutinose, respectively, inan inert solvent such as chloroform or methylene chloride, underanhydrous conditions.

The mixture of O-(hexaacetyl α- and -β-rutinosyl) trichloroacetimidatesis obtained by the action of hexaacetyl-1-rutinose (obtained by theaction of benzylamine or ammonia on heptaacetorutinose in an aproticsolvent such as acetonitrile, tetrahydrofuran or ethyl ether) ontrichloroacetonitrile, in the presence of a base, for example potassiumcarbonate or sodium hydride, in an aprotic solvent such as methylenechloride, chloroform or diethyl ether and under anhydrous conditions.O-[Hexaacetyl-6-O-(α-L-arabinofuranosyl)-α- and -β-D-glucopyranosyl]trichloroacetimidates are obtained in the same manner.

The second stage consists in the catalytic nucleophilic substitution ofthe leaving group introduced, by paranitrophenol, by a monoterpenol orby an alcohol. Thus, after purification by chromatography on silica gel,a peracetylated β-p-nitrophenyl, β-terpenyl or β-alkyl glycoside isobtained.

Thus, the action of monoterpenols such as geraniol, nerol, α-terpineolor linalool, or of alcohols such as benzyl alcohol or 2-phenylethanol,on peracetyl-α-bromo-D-glucopyranoside or peracetyl-α-bromorutinoside isperformed in the presence of silver carbonate, in an aprotic solventsuch as, for example, ether, methylene chloride or chloroform, in thepresence of drierite or a 0.4 nm (4 Å) molecular sieve; or in thepresence of a soluble catalyst such as mercuric cyanide, inacetonitrile, in the presence of a 0.4 nm (4 Å) molecular sieve; theaction of p-nitrophenol on peracetyl-α-chlororutinoside is obtained inpyridine, in the presence of silver carbonate and drierite; that ofmonoterpenols such as linalool, geraniol or α-terpineol onO-(peracetyl-α- and β-rutinosyl) trichloroacetimidates orO-[hexaacetyl-6-O-(α-L-arabinofuranosyl)-α- andβ-D-glucopyranosyl]trichloroacetimidates is carried out in the presenceof a 0.4 nm (4 Å) molecular sieve and boron trifluoride etherate orpara-toluenesulphonic acid in methylene chloride or chloroform.

Chromatography of the desired peracetyl β-glycosides on silica gel iscarried out by eluting with ether/petroleum ether, chloroform/ether,methylene chloride/ether or ethyl acetate/petroleum ether mixtures.

The final stage consists in the removal of the protective acetyl groupsfrom the sugar portion of the glycosides formed; the deacetylation isperformed by transesterification in methanol in the presence of a basiccatalysis such as sodium methylate.

Among peracetylated saccharides which are the starting materials forthese syntheses, only1,2,3,4-tetra-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-β-D-glucopyranoseis not commercially available. Its preparation may be accomplished bythe synthesis described above, starting with 1,2,3,5-tetra-O-acetyl-α,β-L-arabinofuranose and 1,2,3,4-tetra-O-acetyl-β-D-glucopyranose.However, it is preferable to carry it out by activation of the1,2,3,5-tetra-O-acetyl-α, β-L-arabinofuranose to3,5-di-O-acetyl-l,2-O-[(1-exo- and1-endo-cyano)ethylidene]-β-L-arabinofuranoses using trimethylsilylcyanide in the presence of a Lewis acid such as stannous chloride, andof the 1,2,3,4-tetra-O-acetyl-β-D-glucopyranose by tritylation to1,2,3,4-tetra-O-acetyl-6-O-trityl-β-D-glucopyranose. The glycosylationreaction between these two synthons is performed under rigorouslyanhydrous conditions, in the presence of triphenylcarbonium perchlorateas a catalyst.

It is possible to apply this synthesis to the preparation ofp-nitrophenyl2,3,4-tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-.beta.-D-glucopyranoside,by coupling the same cyanoethylidene derivative with p-nitrophenyl2,3,4-tri-O-acetyl-6-O-trityl-β-D-glucopyranoside. However, thisglycosylation leads, besides the expected diholoside, to the diholosidehaving a 1-4 inter-saccharide bond: their separation is possible bychromatography on silica gel, eluting with an ethyl acetate/petroleumether mixture.

EXAMPLE 1

Decaacetyl rutin

In a 500-ml round-bottomed flask equipped with a condenser with acalcium chloride guard tube, 25 g of rutin are added with magneticstirring to 150 ml of anhydrous pyridine. While the mixture is cooled ina cold water bath, 100 ml of acetic anhydride are then added in thecourse of approximately 10 minutes, and stirring is continued for 24hours. The reaction medium is then poured into 1.5 1 of icecold water:the acetylated derivative precipitates in white crystals. These crystalsare filtered off, washed with water and with a little ethyl ether andthen dried under vacuum in a desiccator over silica gel. 38.3 g areobtained (yield: 99%)-TLC, silica gel-ether Rf=0.22, m.p. 128°-135° C.

EXAMPLE 22,3,4-Tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamno-pyranosyl)-.alpha.-D-glucopyranosylchloride

Approximately 6 g of ZnCl₂ are melted over a Bunsen burner in acrucible. It is allowed to cool under aluminium foil and 4 g of thisZnCl₂, which has been coarsely ground, are rapidly placed in a 250-mlround-bottomed flask equipped with a condenser and provided with acalcium chloride guard tube.

The following are then added with magnetic stirring:

80 ml of anhydrous chloroform, then

20 g of decaacetylated rutin;

finally, approximately 20 ml of 1,1,-dichloromethyl methyl ether areintroduced in the course of 5 minutes;

the mixture is then brought to 75°-77° C. for 2 hours.

The chloroform solution is decanted and the pasty residue is washed fromthe flask with 20 ml of chloroform, and this is combined with thechloroform solution which is evaporated under vacuum in a rotaryevaporator at 35°-45° C.

The oily yellow residue is taken up in 500 ml of ethyl ether which iswashed with ice-cold water, with saturated Na₂ CO₃ solution and thenagain with ice-cold water. The organic phase is dried over Na₂ SO₄ andfiltered, and the solution is concentrated in a rotary evaporator undervacuum at about 35° C. The residue, which has partially crystallized, isrecrystallized in ethyl ether, and then a second time in anhydrousethanol. The white crystals obtained are dried under vacuum in adesiccator over silica gel.

6.3 g are obtained (yield-53%)-TLC, silica gel ether Rf=0.52, m.p.148°-150° C. EXAMPLE 3 p-Nitrophenyl2,3,4-tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-.beta.-D-glucopyranoside

In an Erlenmeyer equipped with a calcium chloride guard tube and amagnetic stirrer, 3 g of drierite and 1.5 g of p-nitrophenol areintroduced into 50 ml of anhydrous pyridine. The mixture is stirred thusfor 1 hour, and 5 g of acetochloro-α-rutinoside and 3 g of silvercarbonate, freshly prepared and dried, are then added.

Stirring is continued in the dark and at room temperature for 24 hours.

The reaction medium is filtered, the precipitate is washed with a littlepyridine and the filtrate is then concentrated in a rotary evaporatorunder vacuum at about 40°-45° C. The residue is taken up twice with 25ml of benzene and concentrated in the same manner to remove the tracesof pyridine.

The residue is again taken up in 100 ml of benzene and the solution iswashed with ice-cold water, N sodium hydroxide solution and then againwith ice-cold water, and finally dried over Na₂ SO₄. After filtration,the filtrate is concentrated in a rotary evaporator under vacuum at35°-40° C. The reddish, pasty residue is purified by chromatography onsilica gel [of a particle size corresponding to passage through a sieveof mesh aperture 67 μm to 199 μm (70 to 230 mesh)], eluting with ethylether-Rf≈0.5. The purest fractions are concentrated in a rotaryevaporator and the white crystals obtained are recrystallized in 95°strength ethanol. 1.4 g are thereby obtained (yield=25%)-TLC, silicagel-ether Rf=0.5, m.p. 185°-187° C.

EXAMPLE 42,3,4-Tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-.alpha.-D-glucopyranosylbromide

1.3 g of heptaacetorutinose and 0.4 ml of acetic anhydride in 20 ml ofchloroform are placed under nitrogen at -4° C. in a 50-ml round-bottomedflask, and 3.8 ml of a 33% strength solution of gaseous hydrobromic acidin acetic acid are added dropwise. Stirring is continued at -4° C. for 2hours, and the reaction medium is then poured into 50 ml of ice-coldwater. The organic phase is separated after settling has taken place,dried over anhydrous sodium sulphate and concentrated in a rotaryevaporator under vacuum at 35° C. The yellow oil obtained is usedwithout purification in the subsequent stage. However, it is possible tocrystallize it by taking it up with a little ethyl ether and leaving itin the cold. On filtration under nitrogen, washing with a little ethylether and petroleum ether and drying in a desiccator in the cold, 310 mgof white crystals are obtained (yield-23%). M.p. 120°-125° C.

EXAMPLE 5 Geranyl2,3,4-tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-.beta.-D-glucopyranoside

In a 50-ml round-bottomed flask, the following are stirred for 24 hoursunder nitrogen at room temperature:

665 mg ofbromo-2,3,4-tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-α-D-glucopyranoside(crude product of the bromination reaction);

1 ml of geraniol;

0.5 g of mercuric cyanide in 10 ml of acetonitrile.

The mixture is then concentrated in a rotary evaporator under vacuum at35° C., and the residue is taken up in 50 ml of ethyl ether. The solidwhich precipitates is filtered off and rinsed with ethyl ether, and thefiltrate is concentrated in a rotary evaporator under vacuum at 35° C.The oily residue is chromatographed on silica gel [of a particle sizecorresponding to passage through a sieve of mesh aperture 67 μm to 199μm (70 to 230 mesh)], eluting successively with ethyl ether/petroleumether (10:90) to remove the excess geraniol and then with ethylether/petroleum ether (75:25) to elute the rutinoside. The fractionscontaining the rutinoside are combined and concentrated at 35° C. undervacuum in a rotary evaporator. 140 mg of a colourless paste, which ithas not been possible to crystallize, are obtained (yield=19%), theproduct being pure in TLC-silica gel; ether/petroleum ether (3:1)Rf=0.33.

EXAMPLE 62,3,4-Tri-O-acetyl-6-O-(2,3,4,tri-O-acetyl-α-L-rhamno-pyranosyl)-D-glucopyranose

In 100-ml round-bottomed flask, a mixture of 600 mg ofheptaacetorutinose and 1.2 ml of benzylamine in 80 ml of ethyl ether areleft with magnetic stirring and under nitrogen for 24 hours at roomtemperature. The reaction medium is then concentrated in a rotaryevaporator under vacuum, and the oily residue is chromatographed on acolumn of silica gel [of a particle size corresponding to passagethrough a sieve of mesh aperture 67 μm to 199 μm (70-230 mesh)], elutingwith ethyl ether. The fractions containing hexaacetylrutinose areconcentrated in a rotary evaporator under vacuum. 500 mg of colourlessoil are obtained (yield=83%); TLC-silica gel-diethyl ether Rf=0.31.

EXAMPLE 7O-[2,3,4-tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamno-pyranosyl)-.alpha.-and -β-D-glucopyranosyl] trichloroacetimidate

2.6 g of2,3,4-tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-D-glucopyranose,1.6 ml of trichloroacetonitrile and 25 ml of methylene chloride aremixed under nitrogen and at room temperature in a 100 ml round-bottomedflask. 1.6 g of anhydrous potassium carbonate are then added withmagnetic stirring, and stirring is continued for 18 hours. The reactionmedium is then filtered and the precipitate is rinsed with 10 ml ofmethylene chloride. The filtrate is concentrated in a rotary evaporatorunder vacuum at 35° C. and the residue is chromatographed on a column ofsilica [of a particle size corresponding to passage through a sieve ofmesh aperture 67 μm to 199 μm (70-230 mesh)], eluting with a 1:1 ethylether/methylene chloride mixture. The fractions containing the imidateare combined and concentrated in a rotary evaporator under vacuum at 35°C. 2.3 g of a colourless oil are obtained (yield=71%); TLC silicagel-diethyl ether Rf=0.76.

EXAMPLE 8 (±)-Linalyl2,3,4-tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-αL-rhamnopyranosyl)-.beta.-D-glucopyranoside

In a 100-ml round-bottomed flask, a mixture of 550 mg of (±)-linalool,650 mg ofO-[2,3,4-tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-.alpha.and -β-D-glucopyranosyl] trichloroacetimidate and 1 g of 0.4 nm (4 Å)molecular sieve in 3 ml of methylene chloride are stirred under nitrogenat room temperature for 30 minutes. 22 μl of a 50% strength solution ofboron trifluoride etherate in methylene chloride are then added, and arealso added after 40 minutes' magnetic stirring. After a further 40minutes' stirring, 650 mg of sodium bicarbonate are added to thereaction medium, which is then washed, volume for volume, with 0.5Maqueous sodium bicarbonate solution and then with water. The organicphase is dried over anhydrous sodium sulphate and concentrated in arotary evaporator under vacuum at 35° C. The oily residue ischromatographed on silica gel [of a particle size corresponding topassage through a sieve of mesh aperture 67 μm to 199 μm (70-230 mesh)],eluting first with a 4:1 petroleum ether/diethyl ether mixture to removethe excess linalool and then with a 1:4 diethyl ether/chloroform mixtureto elute the acetylated heteroside. The fractions containing the latterare combined and concentrated in a rotary evaporator under vacuum at 35°C. 190 mg of a colourless oil are obtained (yield=29%); TLC-silica gel;diethyl ether/petroleum ether (4:1) Rf=0.34.

EXAMPLE 9 Geranyl 6-O-(α-L-rhamnopyranosyl)-β-D-glucopyranoside

In a 50-ml Erlenmeyer swept with a stream of nitrogen, 0.2 g of geranyl2,3,4-tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-.beta.-D-glucopyranosideare dissolved with mechanical stirring in 2 ml of anhydrous methanol.

0.3 ml of a sodium methylate solution (prepared from 230 mg of sodiumand 100 ml of anhydrous methanol) is then added in the course of 30seconds, and stirring is continued under nitrogen in an oil bath at68°-70° C. for 20 minutes. The Erlenmeyer is then cooled in a water/icebath and approximately 0.2 ml of wet Dowex 50W×4(H⁺) [of a particle sizecorresponding to passage through a sieve of mesh aperture 74/147 μm(100/200 mesh)] is added so that the pH of the solution becomes in theregion of 7. The resin is then filtered off and the filtrate isconcentrated in a rotary evaporator under vacuum at 25° C. The oilyresidue obtained is purified by chromatography on a column of silica gel60 [of a particle size corresponding to passage through a sieve of meshaperture 67 μm to 199 μm (70 to 230 mesh)], eluting with a 3:1 ethylacetate/methanol mixture. The fractions containing the geranylβ-rutinoside are combined and concentrated in a rotary evaporator undervacuum at 25° C. 110 mg of a colourless paste, which it has not beenpossible to crystallize, are obtained, the product being pure inTLC-silica gel; ethyl acetate/methanol (3:1) Rf=0.35.

EXAMPLE 102,3,4-Tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabino-furanosyl)-.alpha.-and -β-D-glucopyranoses

Gaseous ammonia is bubbled for 15 minutes into a 100-ml round-bottomedflask containing 25 ml of a 7:3 tetrahydrofuran/methanol mixture andmaintained at 0° C. in crushed ice.

400 mg of1,2,3,4-tetra-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-β-D-glucopyranosideare then added.

The flask is then allowed to return to room temperature while stirringis continued.

The reaction is followed by TLC (silica gel; methylene chloride/ether,6:4). When there is no more starting material (after approximately 15minutes), the reaction is stopped and the medium is concentrated todryness under reduced pressure at 40° C.

The residue is then purified on a column of silica, eluting with a 6:4methylene chloride/ether mixture. 250 mg of product deacetylated at the1-position are thereby obtained (yield: 67%). TLC-silica gel-methylenechloride/ether (6:4); Rf=0.36.

EXAMPLE 11O-[2,3,4-Tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-ara-binofuranosyl-.alpha.and -β-D-glucopyranosyl] trichloroacetimidate

250 mg of2,3,4-tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-.alpha.-and -β-D-glucopyranoses, 2.5 ml of absolute methylene chloride, 0.16 mlof trichloroacetonitrile and 160 mg of anhydrous potassium carbonate areintroduced into a reactor maintained under nitrogen.

The medium is left for 48 hours at room temperature with magneticstirring. It is checked that the reaction is complete by TLC (silicagel; methylene chloride/ether, 6:4).

The medium is then taken up with 10 ml of CH₂ Cl₂ and filtered on asinter. The organic phase is washed successively, volume for volume,with saturated aqueous NaHCO₃ solution and with ice-cold water; it isthen dried over anhydrous sodium sulphate and thereafter concentrated todryness at 35° C. under reduced pressure. 264 mg of a yellowish oil arethereby obtained (yield=88%)-TLC-silica gel methylene chloride/diethylether (6:4)-Rf=0.5.

EXAMPLE 12 Geranyl2,3,4-tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-arabino-furanosyl)-.beta.-D-glucopyranoside

250 mg ofO-[2,3,4-tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-L-arabinofuranosyl)-α- and-β-D-glucopyranosyl] trichloroacetimidates, 250 μl of geraniol and 1.5ml of anhydrous methylene chloride are introduced into a 50-ml reactormaintained under nitrogen.

The medium is maintained under nitrogen and with magnetic stirring; 20μl of a 50% strength solution of boron trifluoride etherate in methylenechloride are added dropwise.

After 3 hours' reaction, it is checked that the reaction is complete byTLC (silica gel, methylene chloride/ether, 7:3), and 50 mg of sodiumbicarbonate are then added to the medium. 10 ml of methylene chlorideare added and the reaction medium is washed, volume for volume, withice-cold 0.5M sodium bicarbonate solution and then with ice-cold water.The organic phase is dried over anhydrous sodium sulphate and taken todryness at 35° C. under reduced pressure. The oily residue is rapidlypurified on a column of silica, eluting first the excess geraniol with a1:1 ether/petroleum ether mixture, and then the product with an 8:2methylene chloride/ether mixture. 150 mg of a colourless oil are therebyobtained (yield=58%). TLC: silica gel, methylene chloride/ether (8:2),Rf=0.49.

EXAMPLE 13 p-Nitrophenyl2,3,4-tri-O-acetyl-6-O-trityl-β-D-glucopyranoside

In a 100-ml round-bottomed flask, 1.42 g of p-nitrophenylβ-D-glucopyranoside and 1.5 g of anhydrous trityl chloride areintroduced into 10 ml of anhydrous pyridine, and the flask is left inthe dark with stirring at 40° C. The reaction is followed by TLC, addingtrityl chloride if necessary. After 24 hours, the flask is brought toroom temperature and 6 ml of acetic anhydride are added. The reactionmedium is left for 48 hours with stirring, the reaction being followedby TLC (silica gel, ether/petroleum ether, 7:3); it is taken up in 500ml of ice-cold water and stirred for 2 hours. The mixture is thenfiltered through celite and the celite is rinsed with dichloromethane.The organic phase is washed successively with ice-cold water, 10%strength hydrochloric acid, saturated aqueous sodium bicarbonatesolution and then ice-cold water. It is dried over sodium sulphate andconcentrated under vacuum at 35° C. The residue is chromatographed on acolumn of silica, eluting with a 7:3 ether/petroleum ether mixture. Thefractions containing the heteroside are combined and concentrated undervacuum at 35° C. 2.6 g of an oil are obtained (yield=83%), the productbeing pure in TLC: silica gel, ether/petroleum ether (7:3), Rf=0.33.

EXAMPLE 14 1,2,3,5-Tetra-O-acetyl-α,β-L-arabinofuranose

A mixture of anhydrous L-arabinose (10 g) and anhydrous methanol (200ml) is treated with 1.06M methanolic hydrochloric acid [prepared byadding acetyl chloride (4.7 ml) and anhydrous methanol (63 ml) at 0°C.]; the mixture is stirred overnight at 0-5° C. Pyridine (40 ml) isadded to neutralize the mixture, which is then concentrated. The residueis taken up several times in pyridine, which is removed by distillation,and it is then dissolved in 80 ml of pyridine. Acetic anhydride (30 ml)is added in the cold state, and the solution is left at room temperaturefor 2 days. Extraction of the reaction medium with ethyl acetate ordichloromethane gives a syrupy product which is then dissolved in amixture of acetic acid (100 ml) and acetic anhydride (25 ml); 5 ml ofconcentrated sulphuric acid are added at 0° C., and the mixture is leftovernight at room temperature. The solution is then immersed in crushedice (150 g) and the mixture is stirred for 2 hours and then extractedwith chloroform. The extract is washed with water and then with aqueoussodium bicarbonate solution; the residue obtained after concentration ofthe organic phase is chromatographed on a column of silica gel (eluant:benzene/ether gradient) and gives1,2,3,5,-tetra-O-acetyl-α,β-L-arabinofuranose in the form of a syrup (18g, 85%), Rf=0.46 (eluant: benzene).

EXAMPLE 15 3,5-Di-O-acetyl-1,2-O-[1-exo- and1-endo-cyano)ethylidene]-β-L-arabinofuranose

Anhydrous stannous chloride (360 mg) and trimethylsilyl cyanide (3 ml)are added to a solution of 1,2,3,5-tetra-O-acetyl-α,β-L-arabinofuranose(3 g) in acetonitrile (10 ml). The mixture is stirred overnight at roomtemperature, then diluted with ether and washed with aqueous sodiumbicarbonate solution (3×75 ml) and then with water. The organic phase isconcentrated and the residue is chromatographed on a column (eluant:benzene/ether gradient) to give the 1-exocyano (994 mg; 37%) and1-endo-cyano (700 mg; 26%) product. Crystallization with ether/pentanegives the 1-exo-cyano isomer (35%), m.p. 66°-69° C., [α]_(D) -6° (C1),Rf=0.56 (eluant: benzene/ether, 3:2). Crystallization with toluene givesthe 1-endo-cyano isomer (23%) m p 107°-110° C. [α]_(D) +51° (C1),Rf=0.37.

EXAMPLE 161,2,3,4-Tetra-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-β-D-glucopyranoseand p-nitrophenyl2,3,4-tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-.beta.-D-glucopyranoside.

Into two round-bottomed flasks closed and joined by a tube shaped like atuning-fork, there are introduced, on the one hand (in one flask) asolution of tritylated glucoside (0.55 mmol) and3,5-di-O-acetyl-1,2-O-[(1-exo- and1-endocyano)ethylidene]-β-L-arabinofuranose (0.5 mmol) in nitromethane(2 ml), and on the other hand (in the other flask) a solution oftriphenylcarbonium perchlorate (0.05 mmol) in 0.2 ml of nitromethane.Both solutions are lyophilized, and then 2 ml of distilled benzene areintroduced into each flask, the contents being lyophilized again; theoperation is repeated a second time; the flasks and the reactants arethus dried for several hours. Dichloromethane (2 ml) is distilled insitu into each of the two flasks. The two solutions are then combinedand left to react overnight at room temperature and in the dark [thelyophilization and also the drying of the reactants, as well as thedistillation of a benzene and dichloromethane over CaH₂, are performedat a pressure of 0.533 Pa (4×10⁻³ mmHg)]. The brilliant yellow reactionmedium is treated with 1 ml of pyridine/water (3:1), and the decolorizedsolution is then diluted with chloroform (50 ml), washed with water(3×30 ml) and concentrated. The residue is purified on a column ofsilica gel, eluting with benzene/ether or ethyl acetate/petroleum ethergradient, to yield:

from 1,2,3,4-tetra-O-acetyl-6-O-trityl-β-D-glucopyranose,

1,2,3,4-tetra-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-β-D-glucopyranose,m.p. 106.5°-108.5° C. (ether/pentane); TLC: silica gel; benzene/ether(3:2), Rf=0.35.

from p-nitrophenyl 2,3,4-tri-O-acetyl-6-O-trityl-β-D-glucopyranoside,p-nitrophenyl2,3,4-tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-.beta.-D-glucopyranoside;TLC: silica gel; ethyl acetate/petroleum ether 1:1, Rf=0.24 andp-nitrophenyl2,3,6-tri-O-acetyl-4-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-.beta.-D-glucopyranoside;TLC, silica gel; ethyl acetate/petroleum ether (1:1), Rf=0.28.

EXAMPLE 17 Geranyl 6-O-(α-L-arabinofuranosyl)-β-D-glucopyranoside

200 mg of geranyl2,3,4-tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-.beta.-D-glucopyranosideare dissolved under nitrogen in 5 ml of anhydrous methanol with magneticstirring. 0.1 ml of a methanolic solution of sodium methylate (preparedfrom 20 mg of sodium and 10 ml of methanol) is added. Stirring iscontinued for 4 hours at room temperature, and the solution is thenneutralized by adding Dowex 50W×4 (H⁺) resin.

The mixture is filtered and the solution is concentrated to dryness in arotary evaporator. The oily residue is purified by chromatography on acolumn of silica gel, eluting with an 8:2 chloroform/methanol mixture toyield 105 mg of geranyl 6-O-(α-L-arabinofuranosyl)-β-D-glucopyranosidein the form of a syrup. Yield 81%, TLC: silica gel, chloroform/methanol(8:2), Rf=0.17.

EXAMPLE 18 p-Nitrophenyl 6-O-(α-L-arabinofuranosyl)-β-D-glucopyranoside

By deacetylation of p-nitrophenyl2,3,4-tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-β-D-glucopyranoside according to the above procedure, p-nitrophenyl6-O-(α-L-arabinofuranosyl)-β-D-glucopyranoside is obtained. TLC: silicagel, ethyl acetate/isopropanol/water (65:30:10), Rf=0.69.

EXAMPLE 19

p-Nitrophenyl 1-β-D-apiose

2 g (6.28 mmol) of 1,2,3,5-tetra-O-acetyl(3')-β-D-apiose and 4 g (28.8mmol) of paranitrophenol in a 100 ml round-bottomed flask are dried bycoevaporation in toluene.

Then 40 mg (0.2 mmol) paratoluene sulfonic acid are added and themixture is heated at 95° C. for 50 minutes under vacuum 10⁻² mm Hg).After cooling, the reactional medium is dissolved in 200 ml CH₂ C₂ andseveral times washed with a saturated solution K₂ CO₃.

The oily residue is dried, concentrated then purified by chromatographyon silica gel 60 column (230-240 Mesh), eluting successively with ethylether/petroleum ether (50/50).

1.4 g of 2,3,5-tri-O-acetyl(3')-O-paranitrophenyl-1-β-D-apiose. M.p.155°-156° C. (yield: 56%) and

0.2 g of 2,3,5-tri-O-acetyl(3')-O-paranitrophenyl-1-α-D-apiose (yield:8%) are obtained.

1.0 of 2,3,5-tri-O-acetyl(3')-O-paranitrohenyl-1-β-D-apiose has beendeacetylated by sodium methylate in anhydrous methanol.

After purification, 0,65 g paranitrophenyl-1-β-D-apiose (yield: 95%) areobtained.

Apiofuranosylglucosides

The general method used to get terpenols and alcoholsapiofuranosylglucosides is the same that the previous one described forthe rhamnosylglucosides and arabinosylglucosides synthesis.

It is possible to apply this synthesis to the preparation of1,2,3,4-tetra-O-acetyl-O-(2,3,5-tri-O-acetyl(3')-β-D-apiofuranosyl)-6-β-D-glucopyranoseby coupling the 3,5-di-O-acetyl(3')-(endo or exo-cyanoethylidene)-1,2-β-D-apiofuranose and the 1,2,3,4-tetra-O-trityl-6-β-D-glucopyranose.

The diholoside heptaacetate is released from the anomeric position ofthe glucose, transformed in its trichloroacetimidate derivate which isthe starting product for respective condensations with:

benzylic alcohol, phenylethylic alcohol, geraniol, nerol,(R)(S)-a-terpineol, (R)-a-terpineol, (R)(S)-β-citronellol,(S)-citronellol, (R)(S)-linalool, (S)-linalool.

Example:

50 mg (0.7 mmol)trichloroacetimidate-1-(2,3,4-tri-O-acetyl-O-(2,3,5-tri-O-acetyl(3')-.beta.-D-apiofuranosyl)-6-O-(aβ)-D-glucopyranose)and 38 ml (8.6 mmol) of (R)(S)-linalool are dissolved in 2 ml anhydrousCH₂ C₂ and a catalytic quantity of BF_(3:) OEt₂ is added. The reactionalmedium is neutralized and extracted by classical methods. Afterpurification and action of sodium methylate, the linalyl diholoside isrecovered with a yield of about 50%.

B. MEASUREMENT OF THE ENZYMATIC ACTIVITIES AND PURIFICATION OF THEENZYMES

1- Measurement of the enzymatic activities

The glycosidase activities are determined by incubating 0.1 ml of 4 mMsubstrate (Glc-pNP, Ara-pNP, Api-pNP, Rha-pNP) in a 100 mM acetatebuffer (pH 4.2) with 0.1 ml of enzyme solution at 40° C. for 20 minutes.The liberation of pNP is estimated by adding 0.6 ml of 1M sodiumcarbonate to the incubation medium and then measuring the opticaldensity at 400 nm. 1 nkat of activity corresponds to the liberation of 1nmol of pNP per second.

Two glycosidases, α-arabinosidase and α-rhamnosidase, were isolated andpurified from commercial preparations containing complex mixtures ofenzymes.

Sweet almond β-glucosidase (Koch-Light, Great Britain, Batch No.2872-01), not displaying any α-arabinosidase, β-apiosidase andα-rhamnosidase type contaminant activity (24 h of incubation at 40° C.,pH 4.2), was used as it is.

The β-apiosidase present in a commercial enzyme preparation (Klerzyme200, Gist-brocades, France) was used without further purification.

2- Purification of the enzymes

2-1. Purification of an α-L-rhamnopyranosidase

The α-rhamnosidase was purified from naringinase (Sigma) rich in theseactivities. The β-glucosidase activities, although low (0.1% of theα-rhamnosidase activity), but capable of liberating glucose over longincubation periods, were removed by the technique of chromatofocusing.

The experimental protocol:

Purification of α-rhamnosidase by chromatofocusing of naringinase on PBE94 gel

50 mg of naringinase, taken up in 4 ml of 25 mM imidazole buffer (pH7.4), are dialysed against the same buffer (25 ml) overnight (5° C.).The dialysate is injected onto a column (1.0×40 cm) of ion exchanger(Polybuffer exchanger PBE 94, Pharmacia, Sweden) equilibrated with thesame buffer. The proteins bound to this gel are eluted by a pH gradientfrom 7.4 to 3.7, created during the migration of Ampholines [Polybuffer74 (Pharmacia) adjusted beforehand to pH 3.7] at a flow rate of 44 ml/h.

After the gradient is completed, the passage of 1M NaCl in 100 mMacetate buffer (pH 3.7) desorbs the proteins retained.

2.8-ml fractions are collected, on which the α-rhamnosidase andβ-glucosidase activities, the pH and the optical density at 280 nm aremeasured.

The chromatographic profile is shown in FIG. 1, to which the legend isas follows:

▪--▪ α-Rhamnosidase activity

□--□ β-Glucosidase activity

∘--∘ Absorbance at 280 nm

+- - - + pH gradient

The α-rhamnosidase activities are eluted in the pH gradient, whereas thewhole of the β-glucosidase activity is retained up to pH 3.7 (pI<3.7)and eluted by the passage of a 1M NaCl solution.

This technique shows that naringinase contains at least 3 (rhamnosidase)isoenzymes of isoelectric points pI 6.2, pI 5.7 and pI<3.7. The second(317 nkat/ml), showing no residual β-glucosidase activity even aftermore than 24 hours' incubation at 40° C., is chosen for the tests ofenzymatic hydrolysis of natural and synthetic glycosides.

The overall recovery yield of the α-rhamnosidase activities isapproximately 62%.

2-2. Purification of an α-L-arabinofuranosidase

Among the commercial enzyme preparations studied, Hemicellulase REG-2(Gist Brocades, France) proved rich in α-arabinosidase activity.Nevertheless, it displays substantial activities of the β-glucosidaseand α-rhamnosidase type. For example, 250 mg of Hemicellulase contain2,327 nkat of α-arabinosidase activity, 2,566 nkat of β-glucosidaseactivity and 236 nkat of α-rhamnosidase activity. Severalchromatographic techniques are applied successively in order to isolateand purify the α-arabinosidase.

Molecular sieving of Hemicellulase on Ultrogel AcA 44 Fractionation

The enzyme solution (250 mg of Hemicellulase in 3 ml of 100 mMcitrate-phosphate buffer, pH 7.2) is dialysed against the same buffer(500 ml) overnight (+5° C.). The dialysate is then placed on a column(1.6×100 cm) of Ultrogel AcA 44 (IBF, France) equilibrated beforehandwith the same buffer. The column is then eluted with the above buffer ata flow rate of 9 ml/h. 1.2-ml fractions are collected and theα-arabinosidase, β-glucosidase and α-rhamnosidase activities, as well asthe optical density at 280 nm, are measured.

Results

The chromatographic profile is shown in FIG. 2, the legend to which isas follows:

-- α-Arabinosidase activity

□--□ β-Glucosidase activity

▪--▪ α-Rhamnosidase activity

∘--∘ Absorbance at 280 nm.

Molecular sieving on Ultrogel AcA 44 enables the two majorα-arabinosidase and β-glucosidase activities to be separated. Theα-rhamnosidase activities are co-eluted with the α-arabinosidaseactivities. The majority of the proteins present in the initial enzymesolution is eluted with the α-arabinosidase activities.

The fraction (52 ml) corresponding to the α-arabinosidase activities(1,750 nkat) also contains trace activities, that is to sayβ-glucosidase (23.4 nkat) and α-rhamnosidase (37.4 nkat), equivalent to1.3% (β-glucosidase) and 2.1% (α-rhamnosidase) relative to theα-arabinosidase activity.

Ion exchange chromatography on DEAE-Sepharose CL-6B of the arabinosidasefraction derived from the molecular sieving Fractionation

The α-arabinosidase-rich fraction (52 ml) was dialysed against 500 ml of25 mM imidazole-HCl buffer (pH 7.5) overnight (+5° C.). The dialysate isthen placed on a column (1.6×40 cm) of DEAE-Sepharose CL-6B (Pharmacia)equilibrated beforehand with the same buffer. The gel is first washedwith this buffer at a flow rate of 108 ml/h. The proteins retained onthe column are then eluted by a linear gradient of sodium chloride (from0 to 0.4M) in the same buffer (imidazole-HCl) at a flow rate of 40 ml/h.4-ml fractions are collected, on which the α-arabinosidase,β-glucosidase and α-rhamnosidase activities and the optical density at280 nm are measured.

Results

The chromatographic profile is shown in FIG. 3, the legend to which isas follows:

-- α-Arabinosidase activity

□--□ β-Glucosidase activity

▪--▪ α-Rhamnosidase activity

∘--∘ Absorbance at 280 nm

- - - - Ionic strength gradient (NaCl)

Under these conditions, the three activities are well separated. Thepeak eluted (69 ml) using 0.3M NaCl and corresponding to theα-arabinosidase activities (365 nkat) now contains only lowβ-glucosidase (0.43 nkat) and α-rhamnosidase (0.32 nkat) activities,equivalent to 0.1% (β-glucosidase) and 0.08% (α-rhamnosidase) relativeto the arabinosidase activity.

It should be noted that, on completion of this stage, theα-arabinosidase fraction contains much less protein than the initialenzyme solution.

This stage enables the α-arabinosidase activity to be purifiedapproximately 14-fold. However, since the presence of low β-glucosidaseand α-rhamnosidase activities can interfer in the hydrolysis of naturaland synthetic glycosides, the purification was refined by theapplication of a further (affinity) chromatographic technique.

Affinity chromatography on concanavalin. A-Ultrogel of theα-arabinosidase fraction derived from the chromatographic stage onDEAE-Sepharose CL-6B Fractionation

The α-arabinosidase fraction (69 ml) is concentrated in a dialysis sackcovered with Sephadex G-200 gel (Pharmacia) to 12 ml. It is dialysedfirst against a 50 mM Tris-HCl buffer (pH 7.2) containing 0.1M NaCl and0.1 mM MnCl₂ overnight (+5° C.), and then injected onto a concanavalinA-UG gel (IBF, France) (1.0×10 cm), equilibrated beforehand with theabove buffer, at a flow rate of 30 ml/h. The column is eluted withmethyl α-D-mannopyranoside (Serva, FRG) in the same buffer, first with alinear gradient (0→0.15M) and then isocratically (0.15M). 1.5-mlfractions are collected, on which the optical density and theα-arabinosidase, glucosidase and α-rhamnosidase activities are measured.The combined fractions displaying α-arabinosidase activities aredialysed against 100 mM acetate buffer (pH 4.2) over-night (+4° C.) toremove the methyl α-D-mannopyranoside.

Results

The chromatographic profile is shown in FIG. 4, the legend to which isas follows:

-- α-Arabinosidase activity

∘--∘ Absorbance at 280 nm

- - - Methyl α-D-mannopyranoside gradient

This stage has made it possible to remove a majority of the proteins andall the residual β-glucosidase and α-rhamnosidase activities, verifiedafter concentration of the fractions by dialysis against dry SephadexG-200 gel. The major α-arabinosidase peak (17 ml; 75 nkat) represents20.5% of the initial activity injected (365 nkat). A part of theactivity (62.5 nkat) trails on elution with methyl mannopyranoside andcorresponds to 17.1% of the initial activity. The enzyme was stronglybound to the gel, and the methyl α-D-mannopyranoside enabled only 37.6%of the initial activity to be eluted (similar results are observed forother enzymes).

As a result of this final stage, the α-arabinosidase activity has beenpurified approximately 27-fold. The enzyme solution used in thehydrolysis tests contains 29.9 nkat/ml of activity.

The numerical results corresponding to the different stages during thepurification of α-arabinosidase from Hemicellulase are collated in Table1.

                                      TABLE 1                                     __________________________________________________________________________    Purification of α-arabinosidase from Hemicellulase REG-2                           Total                                                                             Activity                                                                           Total                                                                              Protein                                                                           Specific                                                                             Puri-                                           Volume                                                                             activity                                                                          yield                                                                              proteins                                                                           yield                                                                             activity                                                                             fication                                  Stage (ml) (nkat)                                                                            (%)  (1) (mg)                                                                           (%) (nkat mg.sup.-1)                                                                     factor                                    __________________________________________________________________________    Crude 3.2  2327                                                                              100  322.6                                                                              100 7.2    1                                         Hemi-                                                                         cellulase                                                                     (250 mg)                                                                      Sieving                                                                             52   1759                                                                              75.6 112.3                                                                              34.8                                                                              15.7   2.2                                       on Ultro-                                                                     gel AcA 44                                                                    Ion   69   365 15.7 3.6  1.1 101.3  14.1                                      exchange                                                                      on DEAE-                                                                      Sepharose                                                                     CL-6B                                                                         Affinity                                                                            34   75  3.2  0.4  0.1 192.3  26.7                                      on Conca-                                                                     navalin                                                                       A-Ulrogel                                                                     __________________________________________________________________________     (1)Proteins are assayed according to Lowry et al. (1951) "Protein             measurement with the Folin phenol reagent", J. Biochem, 193, pages 265-27                                                                              

Properties of the purified α-arabinosidase pH optimum

The enzyme solution is incubated in the presence of its substrate(Ara-pNP) in universal buffer (Britton and Robinson type) of pH varyingfrom 3.0 to 7.0. The activities measurements are carried out at thevarious incubation pH values under the conditions described at thebeginning of this section A. The residual activity as a function of thepH is shown in FIG. 5a (curve --).

The α-arabinosidase activity is maximal at around pH 3.7-4.0.

Stability of the activity as a function of the pH

The enzyme solution is incubated for 50 min at 60° C. in a universalbuffer whose pH varies from 3.0 to 6.5. The samples are then dialysedagainst 11 of 100 mM acetate buffer (pH 4.2) for 5 hours (+5° C.). Theresidual activity measured is shown in FIG. 5a (curve ∘--∘).

The α-arabinosidase activity is relatively stable between pH 3.8 and4.9. This stability decreases rapidly at pH values below 3.5 and above5.5.

Temperature optimum

The α-arabinosidase activity is determined after incubation of thereaction medium at different temperatures varying from 5° C. to 80° C.The relative activity as a function of the incubation temperature is inshown FIG. 5a (curve --).

The activity is maximal at 60° C.

Thermal stability

The enzyme solution is maintained at different temperatures (from 5° C.to 80° C.) in 100 mM acetate buffer (pH 4.2) for 30 minutes: theresidual activity is then measured and the results are shown in FIG. 5b(curve ∘--∘).

The α-arabinosidase activity is stable up to 60° C., and above this thestability decreases abruptly. It is almost totally inactivated after atreatment at 70° C. for 30 minutes.

C. ENZYMATIC HYDROLYSES

The separate or sequential action of four enzymes, anα-L-arabinofuranosidase (E. C. 3.2.1.55, designated α-arabinosidase), anα-L-rhamnopyranosidase (E. C. 3.2.1.40, designated α-rhamnosidase), aβ-D-apiofuranosidase (designated β-apiosidase) and aβ-D-glucopyranosidase (E. C. 3.2.1.21, designated β-glucosidase) wasstudied on different glycosidic substrates. The latter are, on the onehand p-nitrophenyl or geranylα-L-rhamnopyranosyl-(1→6)-β-D-glucopyranosides (designated Rha-Glc-pNPor Rha-Glc-Ger), geranyl β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside(designated Api-Glc-Ger) and p-nitrophenylα-L-arabinofuranosyl-(1→6)-β-D-glucopyranoside (designated Ara-Glc-pNP),and on the other hand a glycosidic extract purified from a muscat must.

It should be noted that the four synthetic glycosides studied possessthe same structures in respect of their carbohydrate portion as thegrape terpene glycosides, but differ in their aglycone which, for threeof them, is p-nitrophenol (pNP).

The enzymatic hydrolysis is followed by thin-layer chromatography (TLC)and by gas chromatography (GC).

Thin-layer chromatography (TLC)

Thin-layer adsorption chromatography was carried out on pieces ofaluminium foil covered with a thin layer (0.2 mm) of silica gel (5553Silica gel 60 without a fluorescence indicator, Merck). An ethylacetate/isopropanol/water (65:30:10 v/v/v) mixture was used as amigration solvent.

The sugars and the glycosides are visualized by means of the followingmixture, prepared immediately before use: 0.2% naphthoresorcinol inethanol/concentrated sulphuric acid (19:1 v/v). The application of thisvisualizing agent is followed by drying the plates in the oven (105° C.)for 15 minutes. Apart from their migration distance, the variouscompounds are also distinguished by their colour. The glycosides arepurple-red, the arabinosides are bluish, the apiosides are green,rhamnose greenish pink, arabinose blue, apiose green and glucose pink.

Moreover, TLC enabled the portion of the glycosidic extract nothydrolysed by the enzymes to be recovered. The zone of thechromatographic plate where the corresponding spot is localized wasscrapped off, the silica gel recovered and suspended in 50 ml ofmethanol. After being left overnight with gentle stirring, thesuspension is filtered on a Buchner funnel and the gel is washed with3×10 ml of methanol. The organic eluates are combined, taken to drynessunder vacuum at 40° C. and then taken up in 500 μl of ultrapure water.The aqueous sample thereby obtained is then ready for acid hydrolysis.

Gas chromatography(GC)

This technique was used in order to analyse both the sugars and theterpene glycosides and also the free terpenols. For this purpose, twodifferent chromatographic systems were employed.

GC of the glycosides

Glycosides and sugars are non-volatile compounds, and are hence notsuited, as such, to an analysis by GC. It is essential to convert themto volatile compounds by means of a selected reagent. Trimethylsilylderivatives were hence formed, according to the following protocol:

40 μl of glycosidic extract and 60 μl of a solution of Glc-pNP (internalstandard) at a concentration of 50 mg/1 in ethyl acetate are introducedinto a suitable flask. The mixture is taken to dryness at 40° C. with astream of nitrogen. 40 μl of the silylating reagent (Tri-sil, Pierce,Rockford, Ill., U.S.A.) are then introduced; the flask is sealed andmaintained at 40° C. for 20 minutes. After rapid cooling, the silylatedsample is then ready for analysis by GC.

The apparatus used consists of a Series 30C gas chromatograph, an"on-column" injector (injected volume 0.5 μl) and a flame ionizationdetector (Girdel, France). A thin film (0.20 μm) of an OV-1 (Girdel)apolar silicone phase is grafted onto the inner wall of the capillarycolumn (50×0.32 mm I.D.). The oven temperature is programmed from 125 to305° C. at the rate of 5° C./minute, then maintained at 305° C. for 15minutes. Finally, the detector temperature is set at 300° C. andhydrogen is used as the carrier gas at a pressure of 120 kPa.

GC of the terpenols

The monoterpene alcohols studied here are sufficiently volatile to bechromatographed as they are. To the pentane extract containing them, theinternal standard, 4-nonanol (for synthesis, Merck, Darmstadt, FRG) isadded in the proportion of 1 μl of a solution at a concentration of 2.89mg/ml in pentane per 50 μl of medium. The mixture is then dried oversodium sulphate, filtered through glass wool and then concentrated to avolume in the region of 100 μl For this purpose, the pentane is firstremoved using a conventional still, and then a Dufton type column of asize suited to the volume remaining to be concentrated.

The constituents of the concentrated extract are separated using acapillary column (25 m×0.32 mm I.D.) containing a CP wax 52 CB(Chrompack, Middelburg, Netherlands) polar phase. The graftedpolyethylene glycol film was selected thick (1.28 μm) in order to permitthe injection of large sample volumes, up to 4 μl. The analysis isperformed using a Fractovap Series 2900 (Carlo Erba, Milan, Italy)chromatograph, equipped with a flame ionization detector maintained at250° C. and an "on-column" injector. Hydrogen was used as the carriergas at a pressure of 60 kPa, and the oven temperature was programmed asfollows: isothermal at 70° C. for 5 minutes, followed by a rise to 195°C. at the rate of 2° C./minute and a plateau at 195° C. for 15 minutes.

High performance liquid chromatography (HPLC)

The benefits of high performance liquid chromatography were exploitedfor isolating some terpene glycosides. For this purpose, fractions werecollected as they emerged from the chromatographic system (moreespecially on elution of the peaks A and B) during the separation of theconstituents of the glycosidic extract. (FIG. 10 below). Fifteensuccessive injections of 20 μl each enabled sufficient material to beisolated for the enzymatic hydrolyses and the analyses by GC to beperformed in order to identify the compounds in question.

The chromatographic system consists of the following components: a Vista5500 chromatograph equipped with a variable wavelength UV/visiblespectrophotometer (Varian Assoc., Sunnyvale, Calif., U.S.A.); a six-wayinjection valve (Valco) equipped with a 20-μl loop; a stainless steelcolumn (220×4 mm I.D.) filled with Spheri-5 (Brownlee Labs., SantaClara, Calif., U.S.A.) octadecyl grafted silica of small particle size(5 μm); as well as a precolumn (37×4 mm I.D.) packed with the samestationary phase.

The chromatography is carried out using a water/--acetonitrileaqueous-organic mobile phase (reversed-phase polarity chromatography)and an elution graduated according to an increase in the acetonitrilecontent from 30 to 40% (by volume) in the course of 10 minutes. Theelution solvent is pumped at a flow rate of 1 ml/min. The detection isperformed at 200 nm and 0.5 absorbance unit full-scale.

1- ENZYMATIC HYDROLYSES ON SYNTHETIC SUBSTRATES

In all the tests relating to the enzymatic hydrolysis of the syntheticsubstrates, the same glycosidase activity (0.15 nkat) is used for eachof the enzymes (α-arabinosidase, apiosidase, α-rhamnosidase,β-glucosidase).

The hydrolysis and analysis protocols are given in plan form (Table 2).

The main abbreviations used will be recalled here:

    ______________________________________                                        pNP        p-nitrophenol                                                      Ara--pNP   p-nitrophenyl α-L-arabinofuranoside                          Rha--pNP   p-nitrophenyl α-L-rhamnopyranoside                           Api--pNP   p-nitrophenyl β-D-apiofuranoside                              Rha--Glc--pNP                                                                 L-rhamnopyranosyl-(1→6)-β-                                                   D-glucopyranoside                                                  Rha--Glc--Ger                                                                 L-rhamnopyranosyl-(1→6)-β-D-                                                 glucopyranoside                                                    Ara--Glc--pNP                                                                            p-nitrophenyl α-L-arabinofuranosyl-(1→6)-β-                 D-glucopyranoside                                                  Ara--Glc--Ner                                                                            neryl  a-L-arabinofuranosyl-(1→6)-β-D-                            glucopyranoside                                                    Ara--Glc--Ger                                                                 L-arabinofuranosyl-(1→6)-β-D-                                                glucopyranoside                                                    Api--Glc--Ger                                                                 D-apiofuranosyl-(1→6)-β-D-                                                   glucopyranoside                                                    Glc--pNP   p-nitrophenyl β-D-glucopyranoside                             Glc--Lin   linalyl β-D-glucopyranoside                                   Glc--Ner   neryl β-D-glucopyranoside                                     Glc--Ger   geranyl β-D-glucopyranoside                                   ______________________________________                                    

                                      TABLE 2                                     __________________________________________________________________________    Plan of the protocol for enzymatic hydrolysis of the synthetic                substrates*                                                                               1st Stage                                                                             2nd Stage                                                 __________________________________________________________________________    200 μl of 4 mM                                                                         Addition of                                                                           Incubation 40° C., 90 min                                                           Addition of 50 μl of a solution of        Ara--Glc--pNP in                                                                          either 50 μl of                                                                    then         β-glucosidase (0.15 nkat) to TEST                                        I                                            100 mM acetate                                                                            a solution of                                                                         12 μl → TLC                                                                      Reincubation 40° C., 90 mins,                                          then                                         buffer (pH 4.2)                                                                           α-arabinosidase                                                                 30 μl → pNP liberated                                                            15 μl → TLC                                    (0.15 nkat)          36 μl → pNP liberated                          (TEST I)                                                                      or 50 μL of a                                                              solution of                                                                   β-glucosidase                                                            (0.15 nkat)                                                                   (TEST II)                                                         200 μl of                                                                              Addition of                                                                           Incubation 40° C., 90 min                                                           Addition of 50 μl of a solution of        Rha--Glc--pNP (4 mM)                                                                      either 50 μl of                                                                    then         β-glucosidase (0.15 nkat) to TEST                                        III                                          or of       a solution of                                                                         extraction with pentane                                                                    Reincubation 40° C., 90 min,                                           then                                         Rha--Glc--Ger                                                                             α-rhamnosidase                                                                  (5 × 250 μl) → GC                                                          extraction with pentane                      (solution not                                                                             (0.15 nkat)                                                                           12 μl → TLC                                                                      (5 × 250 μl) - GC                   titrated) in 100 mM                                                                       (TEST III)                                                                            30 μl → pNP liberated                                                            15 μl - TLC                               acetate buffer                                                                            or 50 μl of       36 μl - pNP liberated                     (pH 4.2)    a solution of                                                                 β-glucosidase                                                            (TEST IV)                                                         __________________________________________________________________________     *The controls are carried out under the same conditions, in the presence      of the inactivated enzymes (95° C., 30 minutes)                   

The results obtained in TLC are shown in FIG. 6, to which the legend isas follows:

Monitoring by TLC of the enzymatic hydrolysis of Ara-Glc-pNP (a),Rha-Glc-pNP (b) and Api-Glc-Ger

1a. Ara-Glc-pNP+β-glucosidase

2a. Ara-Glc-pNP+α-arabinosidase

3a. Ara-Glc-pNP+α-arabinosidase+β-glucosidase

1b. Rha-Glc-pNP+β-glucosidase

2b. Rha-Glc-pNP+α-rhamnosidase

3b. Rha-Glc-pNP+α-rhamnosidase+β-glucosidase

The action of β-glucosidase on the different synthetic substrates givesrise to no modification.

The action of the α-arabinosidase on Ara-Glc-pNP causes thedisappearance of this substrate and the appearance of arabinose andGlc-pNP.

Similarly, the action of α-rhamnosidase on Rha-Glc-pNP or Rha-Glc-Gergives rise to the disappearance of these substrates and the appearanceof rhamnose and Glc-pNP or Glc-Get.

This constitutes the first stage of the hydrolysis.

The consecutive action of β-glucosidase on each of these mediapreviously incubated with α-arabinosidase or α-rhamnosidase leads to thedisappearance of Glc-Ger and Glc-pNP and the appearance of glucose(second stage).

As regards the corresponding aglycones (pNP or geraniol), they areliberated only after the sequential action of both glycosidases:α-rhamnosidase or α-arabinosidase, and then β-glucosidase.

In another aspect, the action of the β-apiosidase (present in Klerzyme200) on Api-Glc-Ger leads to the disappearance of the substrate and theappearance of apiose and glucose.

These results show that the enzymatic hydrolysis of the glycosidesstudied proceeds well with the two stages described above.

2- ENZYMATIC HYDROLYSIS OF A GLYCOSIDIC EXTRACT PROTOCOL FOR PRODUCTIONOF THE GLYCOSIDIC EXTRACT

The glycosidic extract was obtained by extraction of a must of Muscat deFrontignan variety grapes collected at maturity in the vines of theStation Experimentale de Pech Rouge (INRA Gruissan, France), followed byremoval of the free terpenols and sugars.

In view of the large number of experiments to be carried out, it wasessential to have recourse to a supply of glycoside which wasconsistent. To this end, the procedure was performed on a large volume(80 liters) of previously centrifuged and sulphite-treated (50 ppm)must, and the extraction of the glycosidic material was carried outusing 80 grams of active charcoal traditionally used in oenology(charcoal type CXV; Ceca S. A., Velizy-Villacoublay, France). Themixture was kept stirred for 4 hours and then left standing overnight.The charcoal was then recovered by filtration through cellulose filters(porosity 40-50 μm).

In order to remove the majority of the sugars also extracted by thecharcoal, the latter was washed with 4×150 ml of water and filtered on aBuchner funnel. Monitoring by TLC enabled it to be verified that thesugar content decreased at each stage without elution of the glycosidestaking place. The latter were then recovered by washing with 5×200 ml ofacetone. Here too, monitoring by TLC enabled the quantitative elution ofthe glycosidic material to be checked. The acetone was removed byevaporation under vacuum and the sample was taken up in 40 ml ofultrapure water. A more exhaustive purification of the glycosidicextract was then carried out by fractionation on an Amberlite XAD-2(Rohm & Haas Co., Philadelphie, Calif., U.S.A.) organic resin preparedaccording to the following protocol: grinding and sieving of theparticles of size corresponding to a sieve of mesh aperture 175-350 μm(between 80 and 40 mesh), followed by three washes in a Soxhlet withmethanol, acetonitrile and diethyl ether, in that order, for 8 hours foreach solvent. The fractionation process, developed at the Laboratoiredes Aromes et des Substances Naturelies (Aromas and Natural SubstancesLaboratory), has already been described in detail. In this study, it wasapplied twice consecutively, and comprised the following stages:

A resin, suspended in methanol, is poured into a glass column (35×1 cm)terminating in a Teflon tap and a glass wool plug. After settling, theresin layer measures approximately 20 cm. Several 50-ml portions ofmethanol are then passed through, followed by washing with 50 ml ofdiethyl ether and finally equilibration of the resin using 100 ml ofultrapure water. The column is then ready for use.

The 40 ml of glycosidic extract are passed through the column at a flowrate of between 2 and 2.5 ml/minute. The column is then washed with 100ml of water to remove residual sugars, and with 100 ml of pentane inorder to elute the free terpenols bound by the resin, the flow ratebeing the same as above.

The glycosides are then recovered by elution using 100 ml of ethylacetate. The composition of this fraction is studied by TLC, whichenables the absence of free sugars to be verified (in this instance, atthe end of the second fractionation on XAD-2).

The glycoside fraction was taken to dryness under vacuum and then takenup in 18 ml of water. The glycosidic extract used in the chromatographicand enzymatic experiments was thereby obtained.

HYDROLYSIS

The experimental protocols for the enzymatic hydrolysis on theglycosidic extract are collated in Table 3.

                                      TABLE 3                                     __________________________________________________________________________    Plan of the protocol for enzymatic hydrolysis of the glycosidic extract*      1st Stage                  2nd Stage                                          __________________________________________________________________________          Addition of either                                                                     Incubation 40° C., 16 h                                                            ADDITION of 50 μl of a solution of                    50 μl of a                                                                          then        β-glucosidase (0.15 nkat) to                        solution of                                                                            Extraction with pentane                                                                   TESTS I and II                                           α-arabinosidase                                                                  (5 × 250 μl) → GC                                                         Reincubation 40°, 16 h                            (0.15 nkat)          then                                                     (TEST I)                                                                      or 50 μl of a                                                                       50 μl aqueous phase                                                                    Extraction with pentane (5 × 250 μl)                                 → GC                                        200 μl of                                                                        solution of                                                                            silylation → GC                                                                    65 μl aqueous phase → silylation                                    → GC                                        glycosidic                                                                          α-rhamnosidase 15 μl aqueous phase → TLC                extract**                                                                           (0.15 nkat)                                                                   (TEST II)                                                                     OR       12 μl aqueous phase →                                      50 μl of a                                                                          TLC                                                                  solution of                                                                   β-glucosidase                                                            (0.15 nkat)                                                                   (TEST III)                                                              __________________________________________________________________________     *The controls are carried out under the same conditions, in the presence      of the inactivated enzymes (95° C., 30 minutes).                       **Before the enzymatic hydrolyses, the glycosidic extract is washed again     several times with pentane in order to remove traces of terpenols.       

This hydrolysis is monitored by TLC and GC. Monitoring of the hydrolysisby TLC

The results are shown in FIG. 7: Monitoring by TLC of the enzymatichydrolysis of the glycosidic extract.

1. Glycosidic extract

2. Glycosidic extract+β-glucosidase

3. Glycosidic extract+α-arabinosidase

4. Glycosidic extract+α-rhamnosidase

5. Glycosidic extract+α-arabinosidase+β-glucosidase

6. Glycosidic extract+α-rhamnosidase+β-glucosidase

The initial glycosidic extract shows a major spot (Rf 0.71, No. 2 onTLC) and two minor spots (Rf 0.67 and 0.79, No. 1 and No. 3 on TLC).

The hydrolysis of this extract with β-glucosidase gives rise to a faintspot having the same Rf (0.26) as glucose, and reduces the larger spot(No. 3).

In the case of the hydrolysis with α-arabinosidase, a substantialreduction is observed in the major spot (No. 2), and the appearance isnoted of spots having Rf values (0.76, 0.79) identical to those of theterpene monoglucosides and with the Rf (0.31) of arabinose, and of afaint unknown spot with an Rf (0.55) less than that of rhamnose.

When the glycosidic extract is subjected to the action ofα-rhamnosidase, the spot No. 1 corresponding to the Rf of Rha-Glc-Gerdisappears and a faint spot with the Rf of the terpene monoglucosidesappears. In this case, only rhamnose (Rf 0.59) is to be found as themonosaccharide liberated.

The consecutive action of β-glucosidase on each of the media incubatedwith either α-arabinosidase or α-rhamnosidase substantially reduces thespots with the Rf of monoglucosides, and gives rise to glucose.

However, since the major spot (No. 2) of the glycosidic extract did notcompletely disappear after sequential action of the glycosidases, theremaining portion was recovered and then subjected to an acidhydrolysis, under the conditions described below. Its hydrolysis at pH3.0 neither gave rise to modification (verified by TLC). On the otherhand, complete hydrolysis with 2M trifluoroacetic acid caused this spotto disappear and liberated glucose, rhamnose and an unknown compound (Rf0.55).

These TLC tests provided information about the fate of the carbohydrateportion of the glycosides during their hydrolysis with purified enzymes.It emerges from these results that the hydrolysis of terpene glycosidesinvolves a sequential hydrolysis. The mechanism of this hydrolysis wasdemonstrated more precisely by GC analysis. Monitoring of the hydrolysisby GC

At each stage of the hydrolysis, the reaction products (sugars, terpeneglycosides and terpenols) are analysed by GC. The results are shown inFIGS. 8 and 9:

FIG. 8: Monitoring by GC of the enzymatic hydrolysis of the glycosidicextract.

a. Silylated glycosidic extract

b. Glycosidic extract+α-arabinosidase

c. Glycosidic extract+α-arabinosidase+β-glucosidase

I.S. Internal Standard (Glc-pNP)

1 and 2. α- and β-arabinose

5 and 6. α- and β-glucose

a. Glc-Lin

b. Glc-Ner

c. Glc-Ger

A. Ara-Glc-Ner

B. Ara-Glc-Ger

FIG. 9: As FIG. 8, in the case of the sequential hydrolysis byα-rhamnosidase+β-glucosidase.

3 and 4. α- and β-rhamnose

The initial (silylated) glycosidic extract contains neithermonosaccharides nor terpene monoglucosides (FIG. 8a).

The action of β-glucosidase does not substantially modify the profile ofthis extract, apart from the appearance of glucose. The latter, alsoobserved by TLC during the monitoring of the hydrolysis of theglycosidic extract by this enzyme, hence probably does not originatefrom the hydrolysis of the terpene precursors, but from otherglycosides.

The action of α-arabinosidase or of α-rhamnosidase gives rise tosubstantial modifications in respect of the profile of the glycosidicextract (FIGS. 8b and 9b). In particular, the two major peaks which havebeen identified (peak A=Ara-Glc-Ner; peak B=Glc-Ger) disappearcompletely after incubation of the medium with α-arabinosidase, whilearabinose appears. The peak C, corresponding to the retention time ofRha-Glc-Ger, decreases after the action of α-rhamnosidase; theappearance of rhamnose is observed simultaneously.

Incubation of the glycosidic extract with each of these twoglycosidases, gives rise, moreover, to three abundant monoglucosides(linalyl, neryl and geranyl glucosides), identified by comparison withreference substances. The larger part (77%) of these terpenemonoglucosides is liberated by the action of α-arabinosidase, theremainder (23%) produced by the action of α-rhamnosidase (Table 4).

                  TABLE 4                                                         ______________________________________                                        Terpene monoglucosides liberated.sup.(1) by α-arabin-                   osidase and α-rhamnosidase from a glycosidic extract                                                  % of mono-                                             α-arabinosidase                                                                   α-rhamnosidase                                                                       glucosides                                      ______________________________________                                        Glc--Lin 10          9            19                                          Glc--Ner 28          5            33                                          Glc--Ger 39          9            48                                          % of mono-                                                                             77          23           100                                         glucosides                                                                    ______________________________________                                         .sup.(1) The results are expressed as a percentage of the total amount of     each compound liberated by the two enzymes.                              

The amounts of terpenols detected during this first stage of thehydrolysis, namely the hydrolysis of the glycosidic extract with each ofthe three glycosidases alone, were only negligible.

The consecutive action of β-glucosidase on the glycosidic extractspreviously incubated either with α-arabinosidase or α-rhamnosidase(second stage) gives rise to the disappearance of the neryl and geranylmonoglucosides and a reduction in the linanyl monoglucoside, and theproduction of glucose (FIGS. 8c and 9c) and terpenols in large amounts.

It is known that the β-glucosidase used, which is extracted from sweetalmond and selected for the purpose of studying the sequentialhydrolysis of terpene glycosides, has a low affinity with respect tolinalyl glucoside, resulting in incomplete hydrolysis of the latterunder the conditions used.

The most abundant of the terpenols liberated are geraniol, nerol andlinalool, in that order. Approximately 80% of these terpenols arederived from the conjugate action of α-arabinosidase an β-glucosidase.

Other terpenols or volatile compounds have also been detected, but insmall amounts, after the sequential action of the enzymes, such asα-terpineol, citronellol, hydroxylinalools, linalool oxides (with thecis and trans furan configurations and the cis pyran configuration),benzyl and phenyl ethyl alcohols, terpenepolyols, norisoprenoids(3hydroxydamascone, 3-oxo-α-ionol), vinylgaiacol and ethylphenol.

The substantial modifications of the chromatographic profiles observedafter the action of α-arabinosidase on the glycosidic extract led to aninvestigation in greater depth of the preponderant glycosides. For thispurpose, fractions corresponding to the peaks A and B of the HPLCprofile were collected. An aliquot portion was Silylated and thenanalysed by GC. The correspondance between the peaks A and B of the twochromatograms of FIGS. 10 and 8a was thus established.

Legend to FIG. 10:

HPLC profile of the glycosidic extract

A. Ara-Glc-Ner

B. Ara-Glc-Ger

Furthermore, each fraction was subjected to the sequential action ofα-arabinosidase and then β-glucosidase. At the end of each stage andaccording to the procedures described, the incubated medium was analysedby GC in order to test for sugars and glucosides on the one hand, andfor terpenols on the other hand. The results are collated in Table 5.Thus, the peaks A and B were identified, respectively, as neryl andgeranyl α-L-arabinofuranosyl-β-D-glucopyranosides. The identity of thegeranyl glycoside was verified, in addition, by co-injection of theglycosidic extract with the synthetic compound.

                  TABLE 5                                                         ______________________________________                                        Identification of the peaks A and B collected by                              HPLC                                                                          Compounds appearing                                                           after action of                                                                             α-arabinosidase                                           α-arabino-                                                                            and then     Compounds                                          sidase        β-glucosidase                                                                         identified                                         ______________________________________                                        Peak A arabinose  glucose      neryl                                                 +          +            α-L-arabino                                     Glc--Ner   nerol        furanosyl-β-D-                                                           glucopyrano-                                                                  side                                           Peak B arabinose  glucose      geranyl                                               +          +            α-L-arabino                                     Glc--Ger   geraniol     furanosyl-β-D-                                                           glucopyrano-                                                                  side                                           ______________________________________                                    

In another aspect, the action of the β-apiosidase on a glycosidicextract (FIG. 11A) was studied after the action of the Hemicellulasepreparation (containing α-rhamnosidase, α-arabinosidase andβ-glucosidase activities) on this extract. The Hemicellulase preparationeliminated the rhamnosyl- and arabinosylglucosides (FIG. 11B).Afterwards, the apiosylglucosides remaining were subjected to thehydrolytic action of the β-apiosidase and β-glucosidase of Klerzyme 200(FIG. 11C). Noteworthy is the disappearance of the apiosylglucosides andthe appearance of linalyl oxide glucosides, which demonstrate thesequential hydrolysis of the apiosylglucosides. The linalylmonoglucosides were not hydrolyzed, contrary to the geranyl and nerylmonoglucosides. This is attributable to the weak affinity of theβ-glucosidase of Klerzyme 200, vis-a-vis the linalyl glucosides.

These results collectively confirm that the enzymatic hydrolysis ofgrape terpene glycosides involves a sequential mechanism identical tothat demonstrated on the synthetic substrates.

3- ENZYMATIC HYDROLYSIS ON NATURAL MEDIA

The enzymatic hydrolysis of a glycosidic extract incorporated, on theone hand into a natural medium (must or dry wine), and on the other handinto a reference medium (buffer), by the action of commercialpreparations containing the requisite three glycosidases, was studied.

The hydrolysis protocol is as follows:

A juice and a wine of a vine variety not containing terpenols andterpene glycosides, but enriched beforehand with a known amount ofglycosidic extract of muscat, are treated with commercial enzymepreparations. The media, as well as controls, are incubated at 25° C.for 86 hours (see the experimental protocol plan below). They are thenpassed through a column of Amberlite XAD-2 according to the protocolapplied for the production of the glycosidic extract, and the terpenolsare assayed by GC.

    ______________________________________                                        Plan of the experimental protocol                                             ______________________________________                                        50 ml must    +0.5 ml  Hemicellulase(1)                                       (pH 3.4:      +0.5 ml  Naringinase(2) Control                                 sugars 180 g/l)                                                                             +2 ml    2% NaN.sub.3 (3)                                       or                                                                            Dry white wine                                                                              +0.5 ml  Hemicellulase                                          (pH 3.1)      +0.5 ml  Naringinase                                                          +1 ml    Glycosidic extract(4)                                                +2 ml    2% NaN.sub.3                                                         +1 ml    Glycosidic extract                                                            Control                                                50 ml buffer  +2 ml    2% NaN.sub.3                                           (50 mM citrate-                                                                             +0.5 ml  Hemicellulase                                          phosphate)    +0.5 ml  Naringinase                                            pH 3.3                 Reference                                                            +1 ml    Glycosidic extract                                                   +2 ml    2% NaN.sub.3                                           ______________________________________                                         (1)0.5 ml of the solution (at a concentration of 15 mg/ml) of                 Hemicellulase REG2 used possessed 70 nkat of arabinosidase activity, 72       nkat of glucosidase activity and 9 nkat of rhamnosidase activity.             (2)0.5 ml of the solution (at a concentration of 5 mg/ml) of Naringinase      used possessed 78 nkat of rhamnosidase activity and 0.1 nkat of               glucosidase activity.                                                         (3)NaN.sub.3 is used in order to avoid microbial growth.                      (4)The glycosidic extract used is obtained by passing a must of Muscat        d'Alexandrie variety grapes through Amberlite XAD2. The amount of             glycosidic extract (1 ml) added in the tests corresponds to that found in     50 ml of must of Muscat d'Alexandrie variety grapes.                     

Under conditions resembling those found in oeno-logical practice, aliberation of terpenols is observed. This liberation is smaller in thecase of must than in that of wine (Table 6). In terms of the percentageof aroma liberated after 86 hours at 25° C. compared with the amountliberated in a reference medium, this comes to 11% for the must and 28%for the wine. It may be noted that geraniol glycosides are moresatisfactorily hydrolysed than those of linalool and nerol.

It was shown that the inhibition of the β-glucosidase activity by theglucose present in large amounts in the must can explain the lowerefficiency relative to the wine.

The results are more satisfactory in the case of wine, since it is thenpossible, taking into account the preponderance of terpene glycosides,to at least double the free terpene fraction.

                  TABLE 6                                                         ______________________________________                                        Hydrolysis of a glycosidic extract incorporated                               into a must and a wine by commercial enzyme preparations                      Compounds liberated relative to the reference                                 medium(1) (%)                                                                                               Benzyl Total                                    Linalool    Nerol    Geraniol alcohol                                                                              terpenols                                ______________________________________                                        MUST   9        6        15     46     11                                     WINE   14       18       49     50     28                                     ______________________________________                                         (1)Reference: Glycosidic extract + citratephosphate buffer, pH 3.3 +          Hemicellulase REG2 + Naringinase.                                        

ACID HYDROLYSES

In order to detect the possible presence of terpene glycosides ofunknown structure in the (TLC) spot resistant to enzymatic hydrolyses bythe purified glycosidases, acid hydrolyses were applied to the compoundscorresponding to this spot recovered by TLC.

1) The recovered medium (250 μl) is taken to dryness at 40° C. with astream of nitrogen. 250 μl of 25 mM citratephosphate buffer (pH 3.0) areintroduced into the flask, which is then sealed and left at 100° C. for20 minutes. After being cooled, the medium is washed with pentane (5×250μl). Internal standard is added to the pentane extract, which is thenconcentrated and analysed by GC. 25 μl of the aqueous phase are thenplaced on a TLC plate.

2) The recovered medium (250 μl), after being dried as above, is treatedwith 250 μl of 2M trifluoroacetic acid and then left at 120° C. for 75minutes. After being cooled, the medium is subjected to the sameanalyses as above.

4. Technological Application

The action of exogenous glycosidases of fungal origin has been studiedduring vinification of naturally sweet wines and of dry wines of thegrape must (Muscat variety). Two enzymatic preparations, Hemicellulaseand Klerzyme 200 (both produced by Gist-brocades) have been tested.

Experimental protocol

Ripe, sound grapes of the Muscat of Frontignan variety were pressed. Themust was sulfited (5 g/hl SO₂) and centrifuged. This was followed by theaddition of the Hemicellulase enzyme preparation (containing (per litermust) 467 nkat α-arabinosidase, 296 nkat β-glucosidase and 31 nkatα-rhamnosidase) and 10 g/hl of a wine yeast (Saccharomyces cerevisiae,type K1, Institut Cooperatif du Vin, Maurin, France).

The naturally sweet wines are obtained after ending fermentation atmid-fermentation by the addition of alcohol until a final concentrationof approximately of 17% (v/v) alcohol is reached.

For dry wines, vinification is continued until the exhaustion of thesugars.

The activity of the glycosidases were followed during the course of thefermentation. The glycosidases were isolated by precipitation with(NH₄)₂ SO₄ and afterwards were measured by use of the correspondingp-nitrophenyl glycosides.

After storing for 1.5 months at 20°-22° C., the free and bound aromacomponent fractions of each sample were extracted on an Amberlite XAD-2column in 50 ml aliquots, and analyzed via GC.

The exogenous glycosidases were effective in the liberation of aromacomponents in the vinification when there was no residual glucose in themedium (as in the case of dry wines). Thus, the content of terpenols(e.g. nerol, geraniol, citronellol, α-terpineol, hydroxy linalools) aswell as norisoprenoid derivatives (β-damascenone, 3-hydroxy-β-damascone)increased in the dry wine originating from the enzymatically-treatedmust (Table 8). This analytical result was confirmed by taste panel. Allof the tasters (15 in all) preferred the enzymatically-treated dry wine.The tasters showed no preference of the naturally sweet wines, whetherenzymatically treated or not. The wines originating from theenzymatically-treated must were judged to be more aromatic (floral) andmore typical.

In sweet wines originating from enzymatically-treated must, thehydrolysis of terpene diglycosides was not complete and was interruptedwhen only monoglucosides remained (FIG. 12). Noteworthy is theperceptible reduction of the major diglycosides (arabinosylglucosides,peaks 7 and 9) and a corresponding increase of the nerol and geraniolmonoglucosides (peaks 2 and 3). The latter are not hydrolyzed in thenaturally sweet wines due to the presence of glucose (30 g/l) which hasthe effect of inhibiting β-glucosidase (originating from Aspergillusniger) activity. On the other hand, these glucosides were hydrolyzed inthe case of the enzymatically-treated dry wines.

However, a β-glucosidase (originating from the yeast Candida wickerhamiiCBS 2928) has been able to hydrolyze neryl- and geranylmonoglucosides inthe naturally sweet wines at a pH of 3.6 and above. This is due to thepoor stability of the Candida wickerhamii β-glucosidase at a pH below3.6.

At pH 3.6, both neryl- and geranylmonoglucosides present in thenaturally sweet wines were hydrolyzed from 19 % to 45 respectively. AtpH 4.0, two thirds of each glucoside were hydrolyzed.

Contrary to arabinosyl- and rhamnosylglucosides, the apiosylglucosidesare not hydrolyzed (FIG. 12, peaks 8 and 10) by the Hemicellulaseenzymatic preparation. This is attributable to the absence ofβ-apiosidase activity in this preparation.

In another aspect, the action of an enzyme preparation (Klerzyme 200)containing β-apiosidase activity, in addition to α-rhamnosidase,α-arabinosidase and β-glucosidase activity, was studied during thefermentation of dry wines from the Muscat of Frontignan must. Klerzyme200 (containing (per liter must) 2,770 nkat β-glucosidase, 704 nkatβ-apiosidase, 276 nkat α-arabinosidase and 35 nkat α-rhamnosidase) and10 g/hl of a wine yeast (Saccharomyces cerevisiae, type K1) were addedto the must.

The enzymatically treated dry wine was distinguished by the increase ofthe free aroma component fraction (Table 9), as compared with the casewhere the Hemicellulase preparation was used. Moreover, the presence ofβ-apiosidase activity in Klerzyme 200 permitted the hydrolysis ofapiosylglucosides (Table 10). It is noteworthy that the linalylglucoside and linalyl oxide glucosides, contrary to the other glucosidesor glycosides, were not hydrolyzed. This may be explained by the weakactivity of the β-glucosidase in the Klerzyme 200 preparation on thesesubstrates.

Other volatile components such as vinyl, guaiacol and 3-oxo-α-ionol weredetected in low concentrations in the enzymatically-treated wine.

The important increase of 2-phenylethanol in the wine is consequent fromits synthesis by the yeast during the course of the fermentation of themust.

In another aspect, the exogenous glycosidases chosen have been found tobe stable at the acidic pH of the must and of the wine.

                  TABLE 8                                                         ______________________________________                                                                          Dry                                         Components  Initial NSW     NSW   wine  Dry wine                              (μg/l)   Must    *       **    *     **                                    ______________________________________                                        Linalol, furanic                                                                          15      31      34    37    48                                    oxide, trans                                                                  Linalol, furanic                                                                          10      54      47    17    20                                    oxide, cis                                                                    Linalol, pyranic                                                                          347     313     326   366   382                                   oxide, trans                                                                  Linalol pyranic                                                                           90      79      88    100   98                                    oxide, cis                                                                    Linalol     1071    1157    1244  1314  1429                                  HO-trienol  9       169     221   163   142                                   α-terpenol                                                                          40      225     256   333   472                                   Citronellol 29      43      63    65    218                                   Nerol       40      32      61    50    321                                   Geraniol    108     86      92    88    316                                   E-8-hydroxy-linalol                                                                       44      Absent  Absent                                                                              Absent                                                                              230                                   Z-8-hydroxy-linalol                                                                       Absent  Absent  Absent                                                                              Absent                                                                              300                                   3,7-dimethyl-,                                                                            282     188     214   246   241                                   1,7-octanediene-3,6-                                                          diol                                                                          3,7-dimethyl-,                                                                            1003    828     829   803   784                                   1,5-octanediene-                                                              3,7-diol                                                                      7-hydroxy-                                                                    6,7-dihydrolinalol                                                                        17      51      54    59    55                                    Total terpenols                                                                           3105    3256    3529  3641  5056                                  Others                                                                        β-damascenone                                                                        Absent  Absent  Absent                                                                              Absent                                                                              39                                    3-hydroxy-β-                                                                         Absent  Absent  Absent                                                                              Absent                                                                              121                                   damascone                                                                     Benzyl alcohol      38      118   142   73                                    361                                                                           Z-phenylethanol                                                                           101     69203   68405 79574 77346                                 ______________________________________                                         NWS: Neutral Sweet Wine                                                       *Control                                                                      **Added with KLERZYME 200 (Gistbrocades)                                 

                  TABLE 9                                                         ______________________________________                                        Components      Initial   NSW     NSW                                         (μg/l)       Must      *       **                                          ______________________________________                                        Linalol, furanic                                                                              Absent    55      136                                         oxide, trans                                                                  Linalol, furanic                                                                              91        61      91                                          oxide, cis                                                                    Linalol, pyranic                                                                              333       356     389                                         oxide, trans                                                                  Linalol pyranic 85        99      88                                          oxide, cis                                                                    Linalol         1020      959     1089                                        HO-trienol      Absent    90      84                                          α-terpenol                                                                              21        130     196                                         Citronellol     Absent    77      425                                         Nerol           48        53      225                                         Geraniol        173       61      300                                         E-8-hydroxy-linalol                                                                           7         18      735                                         Z-8-hydroxy-linalol                                                                           Absent    37      497                                         3,7-dimethyl-,  190       237     360                                         1,7-octanediene-3,6-diol                                                      3,7-dimethyl-,  1014      1127    1969                                        1,5-octanediene-                                                              3,7-diol                                                                      7-hydroxy-      Absent    68      104                                         6,7-dihydrolinalol                                                            Total terpenols 2982      3428    6671                                        Others                                                                        β-damascenone                                                                            Absent    Absent  62                                          3-hydroxy-β-damascone                                                                    Absent    Absent  57                                          Benzyl alcohol  95        48      174                                         Z-phenylethanol 112       68131   55829                                       ______________________________________                                         NWS: Neutral Sweet Wine                                                       *Control                                                                      **Added with KLERZYME 200 (Gistbrocades)                                 

                  TABLE 10                                                        ______________________________________                                        Composition     Initial    Dry     Dry                                        μg/l         Must       wine*   wine**                                     ______________________________________                                        Linalyl glucoside and                                                                         323        292     429                                        linalyl furanic oxide                                                         Neryl glucoside 58         59      Absent                                     Geranyl glucoside                                                                             67         70      Absent                                     Linalyl rhamnosylglucoside                                                                    174        183     Absent                                     Neryl rhamnosylglucoside                                                                      92         89      Absent                                     Geranyl rhamnosylglucoside                                                                    73         73      Absent                                     Linalyl arabinosylglucoside                                                                   78         79      Absent                                     Neryl arabinosylglucoside                                                                     247        252     Absent                                     Geranyl arabinosylglucoside                                                                   263        231     Absent                                     Neryl apiosylglucoside                                                                        301        293     Absent                                     Geranyl apiosylglucoside                                                                      79         81      Absent                                     Benzyl apiosylglucoside                                                                       87         87      Absent                                     Geranic and neryl acidic                                                                      39         37      Absent                                     glycoside                                                                     ______________________________________                                         *Control                                                                      **Added with KLERZYME 200 (Gistbrocades)                                 

We claim:
 1. A method for obtaining aroma components and aromas fromtheir glycosidic precursors said method comprising:hydrolyzing aglycosidic substrate comprising a β-apioside glycosidic precursor with afirst enzyme composition comprising a β-apiosidase capable of cleaving a1,6-glycosidic bond of said β-apioside glycosidic precursor, whereby atleast one monoglycoside product is obtained; and hydrolyzing saidmonoglycoside product with a second enzyme composition comprising atleast one enzyme capable of cleaving an aglycone-carbohydrate linkagebond, whereby said aroma components and aromas are obtained.
 2. Themethod according to claim 1, wherein said β-apiosidase is aβ-D-apiofuranosidase.
 3. The method according to claim 1, wherein saidβ-apioside glycosidic precursor is a β-D-apiofuranosyl-β-D-glucoside. 4.The method according to claim 1, wherein said hydrolyzing with saidfirst enzyme composition and said hydrolyzing with said second enzymecomposition are simultaneous.
 5. The method according to claim 4,wherein said glycosidic substrate is derived from a vegetable materialselected from the group consisting of (a) a fruit, (b) an aromaticplant, (c) a flowering plant, and (d) in vitro plant cell cultures. 6.The method according to claim 5, wherein said vegetable material isderived from grapes.
 7. The method according to claim 6, wherein saidvegetable material derived from grapes is selected from the groupconsisting of (a) grape juice, (b) wine, (c) a derivative of grape juiceor wine, and (d) a by-product of the vinification of aromatic vinevarieties.
 8. The method according to claim 7, wherein said glycosidicsubstrate is an extract of a must originating from an aromatic vinevariety and wherein said extract is free of terpenols and sugars.
 9. Themethod according to claim 1, wherein said glycosidic substrate is anatural medium.
 10. The method according to claim 9, wherein saidnatural medium is juices and wines of a vine variety with the provisothat when said natural medium does not contain terpenols and terpeneglycosides, said natural medium is enriched with a glycosidic extractderived from a vegetable material derived from grapes selected from thegroup consisting of (a) grape juice, (b) wine, (c) a derivative of grapejuice or wine, and (d) a by-product of the vinification of aromatic vinevarieties.
 11. The method according to claim 1, wherein said glycosidicsubstrate is a synthetic substrate.
 12. The method according to claim11, wherein said synthetic substrate is selected from the groupconsisting of a p-nitrophenylα-L-rhamnopyranosyl-(1→6)-β-D-gluco-pyranoside, a geranylα-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside, and a p-nitrophenylα-L-arabinofuranosyl-(1→6)-β-D-glucopyranoside.
 13. The method accordingto claim 1, wherein said first enzyme composition further comprises anα-arabinosidase.
 14. The method according to claim 13, wherein saidα-arabinosidase is an α-L-arabinofuranosidase (E.C.3.2.1.55).
 15. Themethod according to claim 1, wherein said aroma components and aromasare selected from the group consisting of geraniol, linalool, nerol,α-terpineol, citronellol, a linalool oxide, a hydroxylinalool, phenylethyl alcohol, ethylphenol, benzyl alcohol, 3-hydroxydamascone,3-oxo-α-ionol and vinylgaiacol.
 16. The method according to claim 1,wherein said aroma components and aromas is a non-odoriferous aglycone.17. The method according to claim 16, wherein said non-odoriferousaglycone is paranitrophenol.
 18. The method of claim 1, wherein saidsecond enzyme composition comprises a β-glucosidase.
 19. The methodaccording to claim 1, wherein said first enzyme composition furthercomprises an α-rhamnosidase.